Interferon alpha 2 (IFNα2) is an important glycoprotein cytokine expressed by activated macrophages. The protein has considerable clinical importance as a broad spectrum anti-viral, anti-proliferative and immunomodulating agent. Recombinant and other preparations of INFα2 have been used therapeutically in a variety of cancer and anti-viral indications in man [reviewed in Sen, G. G. and Lengyel P, (1992), J. Biol. Chem. 267: 5017-5020]. Currently there are a number of IFNα preparations in clinical use, including native recombinant IFNα2s produced in E. coli (IFNα2a, RoferonA®, Hoffman-La Roche; IFNα2b, IntronA®, Schering-Plough; IFNα2c, Berofor®, Basotherm) and more recently a synthetic IFNα, also produced in E. coli, based upon the consensus sequence of all subtypes (Infergen®, InterMune).
A major use of IFNα2 is the treatment of chronic hepatitis C virus (HCV) infection. Treatment with IFNα alone results in sustained virus clearance in around 10% of patients, although more recently sustained viral responses of 40% have been achieved with the combination of IFNα2 with ribavirin [Davis G L, et al, (1998) N. Engl. J. Med.; 339:1493-1499; McHutchison J G et al (1998) N. Engl. J Med.; 339:1485-1492; Reichard O, et al (1998) Lancet. 351:83-87]. IFNα therapy is intensive and associated with severe side effects leading to withdrawal of treatment in up to 20% of cases. The rationale for intensive therapy is that IFNα2b has a relatively short serum half-life [Glue P, et al (2000) Clin. Pharmacol. Ther.; 68:556-567], requiring administration by subcutaneous injection once daily or three times weekly for anti-viral efficacy.
The short half-life and frequent dosing have been recognised as problematic in long-term treatment. To address this ‘pegylated’ versions of RoferonA® and IntronA® (Pegasys® and Peg-Intron®) have been introduced and a similar version of Infergen® is in phase II clinical trials. These modified interferons are conjugated to polyethylene glycol moieties which increases the serum half-life 10 to 20 fold (6,7), thereby reducing the dosing frequency to once weekly (180 μg or 1.4 μg/Kg for Peg-Intron™ and Pegasys™ respectively) without adversely affecting clinical efficacy [Glue P. et al (2000) ibid; Perry C M, et al (2001) Drugs; 61:2263-2288; Glue P, et al (2000) Hepatology; 32:647-653]. In these studies, the side effect profiles are similar to unmodified interferon.
Another strategy for increasing serum half-life is to link IFNα to human serum albumin [Osborn B L, et al (2002) J. Pharmacol. Exp. Ther.; 303:540-548]. Albuferon® consists of IFNα linked to the C-terminus of human serum albumin and, in cynomolgus monkeys, has a half-life 3 fold greater than that of pegylated IFNα and 18 fold greater than unmodified IFNα. Data from studies in humans are not yet available for this molecule. However for both pegylated and albumin linked IFNα, the in vitro specific activity of the modified proteins is reduced compared to native protein, to 28% with Peg-Intron® [Grace M, et al. (2001) Cytokine Res.; 21:1103-1115] and to 10% or less with Pegasys® and Albuferon® [Osborn B L, et al (2002) ibid; Bailon P, et al (2001). Bioconjug Chem. 12:195-202].
Despite the significant therapeutic benefit found in using IFNα, resistance to therapy in certain patients has been documented and one important mechanism of resistance has been shown to be the development of neutralising antibodies detectable in the serum of treated patients [Quesada, J. R. et al (1985) J. Clin. Oncology 3:1522-1528; Stein R. G. et al (1988) ibid; Russo, D. et al (1996) Br. J. Haematol.; 94:300-305; Brooks M. G. et al (1989) Gut 30: 1116-1122]. An immune response in these patients is mounted to the therapeutic interferon despite the fact that a molecule of at least identical primary structure is produced endogenously in man Repeated dosing over several months induces anti-IFNα neutralising antibodies in up to 80% of patients, depending upon the indication [Schellekens H, et al (1997) J Interfron Cytokine Res. 17 Suppl 1:S5-8], with the reported frequency for chronic HCV infection ranging from 7% to 60% [Schellekens H, et al (1997) ibid]. Available evidence suggests that patients who develop neutralising antibodies are more likely to fail to respond to treatment and suffer relapse than those who do not develop antibodies [Ross C, et al (2002) J Interferon Cytokine Res.; 22: 421-426; McKenna R. M, et al (1997) J. Interferon Cytokine Res.; 17:141-143; Russo D, et al (1996) ibid; Milella M, et al (1993) Liver; 13:146-150; Primmer O. (1993) Cancer; 71:1828-1834], although in some cases treatment can be rescued by the subsequent use of purified leukocyte interferon [Russo D, et al (1996) ibid; Oberg K, & Aim G. (1997) Biotherapy; 10:1-5; Tefferi A, & Grendahl D. C. (1996) Am. J. Hematol.; 52: 231-233; Milella M, et al (1995) Hepatogastroenterology; 42:201-204].
The reason for the development of antibodies to recombinant IFNα is unclear since the protein is present naturally and expression increases sporadically in response to events such as viral infection. The route and frequency of dosing, the immune modulatory effects of IFNα, and the presence of protein aggregates in the pharmaceutical preparations may all play a role in the breakdown of immune tolerance. However, irrespective of any facilitating factors, the pivotal feature leading to the induction of an immune response is the presence within the protein of peptides that can stimulate the activity of T-cells via presentation on MHC class II molecules. Such peptide sequences are “T-cell epitopes” and are commonly defined as any amino acid residue sequence with the ability to bind to MHC Class II molecules. Implicitly, a “T-cell epitope” means an epitope which when bound to MHC molecules can be recognised by a T-cell receptor (TCR), and which can, at least in principle, cause the activation of these T-cells by engaging a TCR to promote a T-cell response.
From the foregoing there is clearly a continued need for INFα2 analogues with enhanced properties. Desired enhancements include alternative schemes and modalities for the expression and purification of the therapeutic, but also and especially, improvements in the biological properties of the protein. There is a particular need for enhancement of the in vivo characteristics when administered to the human subject. In this regard, it is highly desired to provide INFα2 with reduced or absent potential to induce an immune response and enhanced biological potency in the human subject.
The inventors have previously disclosed the critical regions of the IFNα2 molecule comprising the T-cell epitopes driving the immune responses to this autologous protein and have provided compositions that reduce the effectiveness or wholly eliminate these sequences from being able to act as T-cell epitopes [WO 02/085941]. Such compositions have been achieved by alteration of the amino acid sequence of the IFNα2 protein, for example by substitution, and the present invention is concerned also with IFNα2 molecules in which amino acid substitution and or combinations of substitution have been conducted. However in the present case, new substitutions and combinations of substitutions made confer the surprising property of significantly enhancing the biological activity of the molecule and such an enhancement in combination with substitutions achieving a reduced immunogenic profile for the protein provide for an improved IFNα2 molecule.
Others have provided modified INFα2 and methods of use and include for example U.S. Pat. No. 4,496,537; U.S. Pat. No. 5,972,331; U.S. Pat. No. 5,480,640; U.S. Pat. No. 5,190,751; U.S. Pat. No. 4,959,210; U.S. Pat. No. 5,609,868; U.S. Pat. No. 5,028,422 and others.
U.S. Pat. No. 5,723,125 describes a fusion protein comprising wild-type human IFNα joined via a peptide linker to a human immunoglobulin Fc fragment. The IFNα domain is oriented N-terminal to the Fc domain in the claimed fusion protein.
U.S. Pat. No. 6,204,022 describes IFNα analogues bearing substitutions from WT especially at positions 19, 20, 22, 24 and 27 and characterised by reduced cytotoxicity in a biological assay.
The general category of “human Fc fusion proteins” and suitable vectors for their production have been described previously [U.S. PAT. NO. 5,541,087; U.S. PAT. NO. 5,726,044 Lo et al (1998), Protein Engineering 11:495-500].